Transcript Nucleic Acids Lectures

DNA-based Tools
• ASO (Allele Specific Oligonucleotides)
– Used to detect specific alleles.
– Can determine if a person is homozygous or
heterozygous for a particular allele
Blood sample from a cystic fibrosis carrier
Isolate DNA
Normal allele
Variant allele
C
G
+
C
G
A
T
C
PCR with primers that flank the
region to be examined
A
T
+
Genomic DNA
PCR-amplified DNA
(split the sample,
isolate the PCR products by electtrophoresis
and blot.)
Hybridize with ASO that is
complementary to normal allele
C
C
G
T
A
A
T
G
C
Hybridization
Hybridize with ASO that is
complementary to variant allele
No hybridization
A
No hybridization
hybridization
Person's genotype:
(N: normal allele; A. affected allele)
N/N
N/A
A/A
(normal) (carrier) (affected)
N/N
N/A
A/A
(normal) (carrier) (affected)
PCR product
Hybridization with normal ASO
Hybridization with variant ASO
Figure 3.7b. ASO-based detection of variant alleles: results.. Blood samples from three individuals
analyzed by ASO hybridization as described in figure 3.7a. The homozygous normal individual shows
hybridization only with the normal ASO, the heterozygous individual shows hybridization with both
ASOs and the individual who is homozygous affected shows hybridization with only the variant ASO.
DNA-based Tools
• ASO (Allele Specific Oligonucleotides)
– Good Knowledge of the allele to be tested is required for
distinguishing one allele from another
– Usually used in conjunction with PCR, electrophoresis and DNA
blotting
• Other technologies for detecting specific point
mutations are available
– All suffer from the disadvantage that each test is directed toward a
specific allele. A person affected by a different variant allele will
not be detected as being affected.
DNA-based Tools
DNA Sequencing
• Good for:
– Idnetifying uncommon mutations
• Disadvantages
– Phenotype may not be apparent from genotype
– Mutations in promotor or introns that affect gene expression may be
missed
DNA-based Tools
DNA Chips
• Primarily a research tool for discovery of disease
genes.
Biopsy
Normal Cells
Tumor cells
mRNA
mRNA
red cDNA
green cDNA
Mix and hybridize
with DNA array


Results:


Red spots:
genes that are
under expressed
in the tumor.
Yellow spots:
(most of them)
equally expressed
genes.
Green spots:
genes that are
over expressed
in the tumor.
Figure 1
The same section of the microarray is shown for three independent hybridizations comparing RNA isolated at the 8
hour time-point after serum treatment to RNA from serum-deprived cells. Each microarray contained 9996
elements, including 9804 human cDNAs, representing 8613 different genes. mRNA from serum-deprived cells was
used to prepare cDNA labeled with Cy3-dUTP and mRNA harvested from cells at different times after serum
stimulation was used to prepare cDNA labeled with Cy5-dUTP. The two cDNA probes were mixed and
simultaneously hybridized to the microarray. The image of the subsequent scan shows genes whose mRNAs are
more abundant in the serum-deprived fibroblasts (i.e., suppressed by serum treatment) as green spots and genes
whose mRNAs are more abundant in the serum treated fibroblasts appear as red spots. Yellow spots represent genes
whose expression does not vary significantly between the two samples. The arrows indicate the spots representing
the following genes: 1) Protein disulphide isomerase-related protein P5, 2) Interleukin-8 precursor, 3) EST
AA057170, 4) Vascular endothelial growth factor.
Protein-based Tools
Western Blots
• Good for:
– detecting the presence of a protein (e.g.
Antitrypsin III)
– detecting the presence of an antibody
• Disadvantages:
– Can not determine if protein is active or not (e.g.
antitrypsin III)
– Must have access to tissue where protein is
expressed (e.g. invasive biopsy might be required)
Protein-based Tools
Immuno-histochemistry
• Good for:
– Identifying tissues that express a particular protein
– Showing over- or under- expression of a protein in
a tissue (e.g. her-2 protein in breast cancer
biopsy)
• Disadvantages:
– Can not determine if protein is active or not (e.g.
antitrypsin III)
– Must have access to tissue where protein is
expressed (e.g. invasive biopsy might be required)
Protein-based Tools
Enzyme Assay
• Good for:
– determining the activity of a suspect enzyme
– determining the concentration of a metabolite
• Disadvantages:
– Must have access to tissue where protein is
expressed (e.g. invasive biopsy might be required)
– Protein must be an enzyme or influence an
enzyme activity
– Activity must be stable during isolation of tissue
DNA-based Tools
FISH
(Fluorescence In Situ Hybridization)
• Good for:
– visualization of large chromosomal changes
• Disadvantages:
– Can not be used to detect small genetic changes
DNA-based Tools
PCR
• Good for:
– amplifying the amount of DNA.
– isolating a segment of DNA
• Disadvantages:
– Only semiquantitative
– Linited size of segment to be amplified
DNA-based Tools
PCR
• Examples:
– Diagnosis of infectious disease (e.g. clamydia,
gonorrhea, HIV)
– Detection of a small deletion or insertion (e.g.
the ΔF508 cystic fibrosis allele)
– Amplification of a specific region to obtain
DNA for other tests (e.g. ASO testing)
DNA-based Tools
ASO
(Allele Specific Oligonucleotides)
•Good for:
– Distinguishing between known alleles that have small
differences in DNA sequence (e.g. point mutations, small
deletions or insertions)
•Disadvantages:
– One needs to know the DNA sequences of the normal DNA
and of the allele to be tested.
DNA-based Tools
DNA sequencing
• Good for:
– Idnetifying uncommon mutations
• Disadvantages
– Phenotype may not be apparent from genotype
– Mutations in promotor or introns that affect gene
expression may be missed
DNA-based Tools
DNA Chips
• Currently a research tool for discovery of disease
genes.
• A potentially powerful diagnostic tool
Hybridization
Nucleic acid
Basics
PCR
Electrophoresis
DNA-Protein
interactions
Chromatin
Gene expression
Diagnostic
tools
Most Proteins bind in the Major
Groove of DNA
Major groove is visible here (minor groove
would be seen from the back of the slide)
Minor groove is visible here (major groove
would be seen from the back of the slide)
Important functional groups
located in the major groove
H Hydrogen bond acceptor
H

H

H Hydrogen bond donor VdW VanderWaals interaction
H

H

H

H

Note the positions of the glycosidic bonds with respect to the major and minor grooves
Examples of Amino acid-Base
Interactions
Common DNA-binding motifs
Fig. 16.6 from Mark’s Basic Medical Biochemistry (page 288)
Common DNA-binding motifs
Specific Examples
The examples presented can be viewed via the
“DNA-protein interactions” tutorial on the
Molecular Basis of Medicine Web site